Using Organotypic Tissue Slices as Substrata for the Culture of Dissociated Cells - Methods in Molecular Biology: Extracellular Matrix Protocols - page 240

3. Methods
3.1. Embedding and Slicing Tissue
1. Autoclave or ethanol flame the vibratome tray to sterilize it and then insert it in

the vibratome.

2. Melt the LMP agarose solution, then cool to and keep at 37 ° C in a water bath.

3. Dissect tissue for slicing and store on ice in PBS/glucose.

4. Fill a 35-mm plastic Petri dish or similar container with the agarose and place the

dissected tissue in the dish ( see Note 1 ), being careful not to transfer too
much liquid with it ( see Note 2 ). Place the dish on ice to speed the hardening of
the agarose.

5. Cut a block (sides 1.5 cm in length) containing the tissue out of the agarose in

the dish using a sterile scalpel or razor blade. In another dish, you can further cut
the block so that the tissue is oriented in the desired plane of sectioning and is at
the correct height for the blade when the block is placed in the vibratome tray
( see Note 3 ).

≈

6. Place a drop of adhesive on the dry tray surface and immediately place the agarose

block on the glue in the proper orientation. Once the glue has set ( see Note 4 ), fill
the tray with ice-cold PBS/glucose so that the block is submerged and surround
the tray with an ice bath.

7. Insert a sterile vibratome blade in the holder on the vibratome ( see Note 5 ).

8. Section the tissue into slices 150-200 m thick ( see Note 6 ). The optimum

vibratome settings for speed and vibration of the blade will depend upon the size
and consistency of the particular tissue and should be empirically determined
( see Note 7 ). Once cut, allow the slice to settle to the bottom of the tray and
separate it from any agarose ( see Note 8 ).

µ

3.2. Mounting Slices onto Supports for Culture
1. C

( see Note 9 ) in a culture well plate or on Parafilm™ in a humidified atmosphere.
Rinse well in PBS before use.

Treat the nitrocellulose disks with the concanavalin A solution for 4-18 h at 4 °

2. Place a disk in the vibratome tray and position it near the slice on the bottom of

the tray. Carefully manipulate the tissue slice onto the disk ( see Note 10 ). Mul-
tiple slices can be placed on the disk, if they can fit.

3. Take a sterile disk holder ( see Subheading 2.2.3. ) and, with forceps, hold it on

the tray bottom next to the disk ( see Note 11 ). With another forceps in the other
hand, grab the edge of the disk with mounted slice(s) and place it into the holder
so that the disk forms the bottom of a well ( Fig. 3F and see Note 12 ).

4. Lift the holder and disk out of the tray so that the well they form remains filled

with the PBS/glucose ( see Note 13 ). Place the holder onto the sterile filter paper
or gauze so that the buffer is drawn down, through the nitrocellulose, by capillary
action, and rapidly lift the holder off the paper just before the slice gets dry ( see
Note 14 ).

5. Using forceps, remove the disk from the holder and transfer it to parafilm or a

culture well plate. Add complete medium before the tissue dries out ( ≈ 100 µ L for